Alcoholic fermentation is an anaerobic catabolism of glucose and some other hexoses leading to production of extracellular ethanol and CO2. During the catabolic process, ATP is formed and used for anabolic purposes resulting in cell growth and propagation (Bai et al., 2008). The efficiency of ATP synthesis during anaerobic glucose catabolism is quite low, varying, depending on the particular glucose catabolic pathway, from one mole of ATP (Entner-Doudoroff or ED pathway) to two moles of ATP (Embden-Meyerhof-Parnas or EMP pathway) per mole of consumed glucose, in contrast to aerobic catabolism where one mole of glucose results in production of 36 moles of ATP. Correspondingly, the efficiency of glucose conversion to cellular biomass by cell division and growth is lowest during anaerobic ED catabolic pathway and is highest during aerobic glucose oxidation.
On a volume basis, alcoholic fermentation represents one of the largest fields of industrial biotechnology being used for production of traditional alcoholic beverages (wine, beer, strong alcoholic beverages, etc.) as well as industrial and fuel ethanol. Due to economic and environmental reasons, an exponential growth in production of fuel ethanol occurred during the last decade (Schubert, 2006). Although lignocellulose is considered to be the most promising feedstock for production of fuel ethanol in the future, current industrial production of fuel ethanol is based on fermentation of traditional feedstocks such as sucrose (of sugarcane or sugar beet) and glucose obtained from starchy materials (corn, potatoes etc). The only organism currently used for industrial ethanol production is the baker's yeast Saccharomyces cerevisiae. This yeast catabolizes glucose through the glycolytic EMP pathway yielding 2 moles of ATP per mole of consumed glucose. Because the metabolic efficiency of this pathway is low, the maximal biomass yield is only about 7% while the ethanol yield from glucose is between 90 and 93% of the theoretical value (Ingledew, 1999). Nonetheless, at the industrial scale, 7% of biomass is a huge amount of by-product, which although possessing some economic value as an animal feed ingredient, still significantly lowers the potential yield of the primary product, i.e., of ethanol that could theoretically be obtained. As annual ethanol production reaches over 50 billion liters, an increase in ethanol yield as little as 1-2% could provide additional hundreds of millions to a billion liters of ethanol and significantly improve economic parameters of ethanol production.
In contrast to S. cerevisiae, the bacterium Zymomonas mobilis ferments glucose through the ED pathway, which gives only 1 mole of ATP per mole of glucose, and directs only 3% of glucose to biomass achieving ethanol yield up to 97% of the theoretically possible value (Sprenger, 1996). Furthermore, Z. mobilis has another important advantage: it is much faster at fermenting glucose to ethanol compared to S. cerevisiae (Sprenger, 1996, Panesar et al., 2006), however, this peculiarity mostly is explained by a faster rate of sugar uptake and subsequent catabolism rather than a lower ATP yield. Attempts to substitute S. cerevisiae by Z. mobilis for production of industrial ethanol were considered as a possible way to increase the ethanol yield by ferementation some 3-4%, which would translate into a hundred million liters in world scale annually. However, Z. mobilis is not free of serious drawbacks which hamper its industrial use now and in near future. They are: (i) it has a very narrow substrate range (sucrose is hardly fermented, and then with low yields), (ii) it has natural auxotrophy for lysine, methionine and some vitamins, (iii) it has non-GRAS status, which prevents use of the biomass by-product as a feed additive (Jeffries, 2005; Bai et al., 2008). Moreover as the main workhorse for ethanol production, the technology of yeast cells for alcoholic fermentation is well developed whereas the fermentation technology for bacterial cells like Z. mobilis is far less developed.
One prior art approach attempted substitution of the essential components of the EMP pathway in yeast with those of the ED pathway from bacteria possessing genes of this pathway such as Z. mobilis. This approach included expression of ED dehydratase and ED aldolase genes edd and eda in a phosphofructokinase deficient mutant of S. cerevisiae (Lancashire et al., 1998). The resulting yeast transformants grew and fermented glucose to ethanol, though the activities of ED dehydratase and ED aldolase were not measured. Apparently this approach did not see further development in the scientific literature. One explanation may be that quite often prokaryotic enzymes display low or no activity in S. cerevisiae hosts (Hahn-Hagerdal et al., 2007), probably due to improper folding or instability. In addition, there may be difficulties in NADP regeneration in yeast engineered to use the ED pathway because NADPH produced by glucose-6-phosphate dehydrogenase should be reoxidized by the alcohol dehydrogenase reaction. However, the major alcohol dehydrogenases in S. cerevisiae use NADH but not NADPH and yeast do not possess NADH/NADPH transhydrogenase (Lescovac et al., 2002; Jeffries and Jin, 2004).
Therefore, there is a continuing need in the art to find ways to enhance ethanol production with yeast such as S. cerevisiae. The present invention provides methods for simultaneously decreasing biomasss accumulation and increasing ethanol production by yeast by manipulation of ATP levels in the yeast.